Translocator Protein (TSPO) Giovanni Natile and Nunzio Denora www.mdpi.com/journal/ijms Edited by Printed Edition of the Special Issue Published in IJMS International Journal of Molecular Sciences Books MDPI Translocator Protein (TSPO) Special Issue Editors Giovanni Natile Nunzio Denora MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Books MDPI Special Issue Editors Giovanni Natile Nunzio Denora University of Bari “Aldo Moro” University of Bari “Aldo Moro” Italy Italy Editorial Office MDPI AG St. Alban-Anlage 66 Basel, Switzerland This edition is a reprint of the Special Issue published online in the open access journal International Journal of Molecular Sciences (ISSN 1422-0067) from 2016–2017 (available at: http://www.mdpi.com/journal/ijms/special_issues/tspo). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: Lastname, F.M. , Lastname, F.M. Article title. Journal Name Year Article number , page range. First Edition 2018 ISBN 978-3-03842-757-5 (Pbk) ISBN 978-3-03842-758-2 (PDF) Articles in this volume are Open Access and distributed under the Creative Commons Attribution license (CC BY), which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book taken as a whole is © 2018 MDPI, Basel, Switzerland, distributed under the terms and conditions of the Creative Commons license CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). Books MDPI Table of Contents About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Nunzio Denora and Giovanni Natile An Updated View of Translocator Protein (TSPO) doi: 10.3390/ijms18122640 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Anna M. Giudetti, Eleonora Stanca, Luisa Siculella, Gabriele V. Gnoni and Fabrizio Damiano Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism doi: 10.3390/ijms17060817 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Leo Veenman, Alex Vainshtein, Nasra Yasin, Maya Azrad and Moshe Gavish Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands doi: 10.3390/ijms17060880 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Valentino Laquintana, Nunzio Denora, Annalisa Cutrignelli, Mara Perrone, Rosa Maria Iacobazzi, Cosimo Annese, Antonio Lopalco, Angela Assunta Lopedota and Massimo Franco TSPO Ligand-Methotrexate Prodrug Conjugates: Design, Synthesis, and Biological Evaluation doi: 10.3390/ijms17060967 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Salvatore Savino, Nunzio Denora, Rosa Maria Iacobazzi, Letizia Porcelli, Amalia Azzariti, Giovanni Natile and Nicola Margiotta Synthesis, Characterization, and Cytotoxicity of the First Oxaliplatin Pt(IV) Derivative Having a TSPO Ligand in the Axial Position doi: 10.3390/ijms17071010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Eleonora Da Pozzo, Chiara Giacomelli, Barbara Costa, Chiara Cavallini, Sabrina Taliani, Elisabetta Barresi, Federico Da Settimo and Claudia Martini TSPO PIGA Ligands Promote Neurosteroidogenesis and Human Astrocyte Well-Being doi: 10.3390/ijms17071028 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Ji Young Choi, Rosa Maria Iacobazzi, Mara Perrone, Nicola Margiotta, Annalisa Cutrignelli, Jae Ho Jung, Do Dam Park, Byung Seok Moon, Nunzio Denora, Sang Eun Kim and Byung Chul Lee Synthesis and Evaluation of Tricarbonyl 99m Tc-Labeled 2-(4-Chloro)phenyl-imidazo[1,2- a ] pyridine Analogs as Novel SPECT Imaging Radiotracer for TSPO-Rich Cancer doi: 10.3390/ijms17071085 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Gurpreet Manku and Martine Culty Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells doi: 10.3390/ijms17091486 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Tamara Azarashvili, Olga Krestinina, Yulia Baburina, Irina Odinokova, Vladimir Akatov, Igor Beletsky, John Lemasters and Vassilios Papadopoulos Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria doi: 10.3390/ijms17122096 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 iii Books MDPI Nasra Yasin, Leo Veenman, Sukhdev Singh, Maya Azrad, Julia Bode, Alex Vainshtein, Beatriz Caballero, Ilan Marek and Moshe Gavish Classical and Novel TSPO Ligands for the Mitochondrial TSPO Can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling doi: 10.3390/ijms18040786 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 iv Books MDPI v About the Special Issue Editors Giovanni Natile received the Laurea in Chemistry at the University of Padua in 1968. After postdoctoral positions at the University College London (1969–1970) and University of Padua (1971–1972), he was appointed Research Assistant and Lecturer at the University of Venice (1972–1980) and then Professor of Inorganic Chemistry at the University of Bari (since 1980). Professor Natile’s main research interests are in the field of Organometallic and Medicinal Bioinorganic Chemistry. (i) In the field of Organometallic Chemistry, he has been interested in the activation of unsaturated molecules and formation of C–C, C–N, and C–O bonds. In this context, he initiated the synthesis of a large series of unprecedented five- coordinate complexes of platinum(II) containing olefins, which can evolve into 4-coordinate superelectrophilic species. (ii) In the field of Bioinorganic Chemistry, he has extensively investigated non- classical antitumor-active platinum compounds demonstrating that also compounds with trans-geometry can have high biological activity. (iii) More recently, his research has been devoted to the targeting and delivery of antitumoral platinum complexes and to the molecular understanding of their uptake and transport mechanisms. Giovanni Natile is co-author of more than 350 papers, international patents and books. He serves as an associate editor of the journal of Bioinorganic Chemistry and Applications and is member of the Editorial Board of the Journal of Inorganic Biochemistry and of the International Journal of Molecular Sciences. He has served as President of the European Association for Chemical and Molecular Sciences “EuCheMS” (2006–2008) and of the Italian Chemical Society (2002–2004) and has been a member of the Management Committee of the European COST ACTIONS D1, D8, D20, D39 and CM1105. Nunzio Denora graduated in Chemistry/Pharmaceutical Chemistry at the University of Bari in 2001. After a PhD in Pharmaceutical Technology (2001–2004) at the University of Bari, he became postdoctoral researcher at the Pharmaceutical Chemistry Department, the University of Kansas (2005). From March 2006, he has been Assistant Professor (full time, permanent position) in Pharmaceutical Technology at the Department of Pharmacy—Drug Sciences, University of Bari Aldo Moro. His research interests concern the following: new prodrugs and conjugates of neurotransmitters useful to overcome the blood–brain barrier; biocompatible polymers and nanocarriers useful to improve the delivery of bioactive drugs; the role of translocator protein in the selective delivery of bioactive drugs to pathologies overexpressing the TSPO. Nunzio Denora is co-author of more than 80 papers, international patents and books. Books MDPI Books MDPI International Journal of Molecular Sciences Editorial An Updated View of Translocator Protein (TSPO) Nunzio Denora 1, * and Giovanni Natile 2, * 1 Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, 70125 Bari, Italy 2 Department of Chemistry, University of Bari “Aldo Moro”, 70125 Bari, Italy * Correspondence: nunzio.denora@uniba.it (N.D.); giovanni.natile@uniba.it (G.N.); Tel.: +39-080-544-2767 (N.D.); +39-080-544-2774 (G.N.) Received: 13 November 2017; Accepted: 4 December 2017; Published: 6 December 2017 Abstract: Decades of study on the role of mitochondria in living cells have evidenced the importance of the 18 kDa mitochondrial translocator protein (TSPO), first discovered in the 1977 as an alternative binding site for the benzodiazepine diazepam in the kidneys. This protein participates in a variety of cellular functions, including cholesterol transport, steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and cell proliferation. Thus, TSPO has become an extremely attractive subcellular target for the early detection of disease states that involve the overexpression of this protein and the selective mitochondrial drug delivery. This special issue was programmed with the aim of summarizing the latest findings about the role of TSPO in eukaryotic cells and as a potential subcellular target of diagnostics or therapeutics. A total of 9 papers have been accepted for publication in this issue, in particular, 2 reviews and 7 primary data manuscripts, overall describing the main advances in this field. Keywords: translocator protein (TSPO); neuroinflammation; steroidogenesis; subcellular targeting 1. Introduction In eukaryotic cells mitochondria play a vital role as they are involved in the control of oxidative phosphorylation and ATP synthesis and in the regulation of apoptosis and translocation of pro-apoptotic proteins from the mitochondrial intermembrane space to the cytosol [ 1 ]. Hence, it is evident that mitochondria can regulate cell survival and that mitochondrial dysfunctions are involved in the onset of several human pathological conditions. For this reason, mitochondria can be considered a potential target for the delivery of therapeutics, although its intracellular localization makes it difficult to reach. Decades of study on the role of mitochondria in living cells have evidenced, in particular, the importance of the 18 kDa mitochondrial translocator protein (TSPO), first discovered in the 1977 as an alternative binding site for the benzodiazepine diazepam in the kidneys. This protein participates in a variety of cellular functions, including cholesterol transport, steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and cell proliferation [ 2 – 7 ]. In accordance with TSPO’s diverse functions, changes in TSPO expression have been linked to multiple diseases, from cancer to endocrine and neurological diseases. Thus, TSPO has become an extremely attractive subcellular target for (1) early detection of disease states that involve the overexpression of this protein [ 8 – 10 ] and (2) selective mitochondrial drug delivery [ 11 – 14 ]. To date, several studies have been carried out on the synthesis of new structurally diverse TSPO ligands and on the preparation of nanosystems or metal complexes that can be directed to TSPO for diagnosis or therapy [ 15 , 16 ], thus highlighting the great interest of the scientific community in understanding the functions of this translocator protein in both normal and pathological conditions. Investigation of the functions of this protein, both in vitro and in vivo , has been mainly carried out using high-affinity ligands, such as isoquinoline carboxamides (e.g., PK 11195) and benzodiazepines (e.g., Ro5-4864). For instance, PK 11195 and Ro5-4864 have been used to explore TSPO distribution Int. J. Mol. Sci. 2017 , 18 , 2640 1 www.mdpi.com/journal/ijms Books MDPI Int. J. Mol. Sci. 2017 , 18 , 2640 and function in various tissues and pathologies, thus allowing the mapping of the “peripheral binding site” in almost every tissue examined [17]. As a result of the great interest in the role of TSPO and its potential use as subcellular target, this special issue, entitled, “Translocator Protein (TSPO)”, was programmed to consolidate new knowledge focused specifically on this receptor. A total of 9 papers were accepted for publication in this issue: 2 reviews and 7 primary data manuscripts, focusing on (1) new functions attributable to TSPO; (2) new potent and selective TSPO ligands; (3) the use of ligands as imaging tools for the early diagnosis of diseases characterized by a high expression of TSPO, such as neuroinflammation and TSPO-rich cancers; (4) TSPO targeted nanocarriers that deliver therapeutics and diagnostics; (5) TSPO ligands that could be used to prepare coordination complexes of metallodrugs for use in diagnosis and therapy; (6) TSPO ligands as pro-apoptotic agents that are potentially useful for the treatment of cancers, and, finally; (7) in vitro and in vivo investigations of the ability of TSPO ligands to affect steroidogenesis. 2. Articles in This Special Issue Mitochondria are involved in several metabolic processes, comprising the energy transduction mechanism which requires the transport of specific metabolites across the inner membrane which is achieved through mitochondrial carriers (MCs), a family of nuclear-encoded proteins sharing several structural features [ 18 ]. The MCs’ function is to facilitate the exchange of metabolites through the inner mitochondrial membrane (IMM). In this regard, Damiano et al. contributed to this special issue with a review concerning two mitochondrial carriers, such as citrate and carnitine/acylcarnitine transporters, involved in fatty acid metabolism [ 19 ]. Interesting new research on the mechanisms involved in the regulation of lipid metabolism in the cell could be sparked from this study. The study of Gavish and coworkers, also reported in this issue [ 20 ], provides a deeper understanding of the overall biological function of TSPO. By applying the microarray analysis of the gene expression in U118MG glioblastoma cells, they discovered that the classical TSPO ligand PK 11,195 can modulate gene expression in this tumor cell line and induce cell morphological changes. In particular, at exposure times of 15, 30, 45, and 60 min, as well as 3 and 24 h, to PK 11,195, changes in gene expression might be associated with several cellular functions including viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was supported microscopically by cell migration, cell accumulation, adhesion, and neuronal differentiation. The authors propose that the modulation in gene expression occurs via mitochondria-to-nucleus signaling; thus, TSPO modulates not only local mitochondrial functions but also nuclear gene expression. Furthermore, Gavish and coworkers identified a novel TSPO ligand, the 2-Cl-MGV-1, able to modulate gene expression of immediate early genes and transcription factors [ 20 ]. The results reported in this work highlight the possible effects on cellular and organismal functions induced by TSPO ligands possibly via modulation of nuclear genes expression promoted by mitochondrial TSPO. This type of modulation can influence several vital cell functions, with major implications on the whole organism in health and disease states. The article of Culty and coworkers in this issue gives further insight into the role of TSPO in living cells [ 21 ]. They showed that TSPO is downregulated during gonocyte differentiation, which is indicative of a possible repressive role. Moreover, expression studies in human normal testes confirmed that TSPO is expressed in subsets of adult germ cells, suggesting a function in acrosome formation, while the analysis of tumor samples revealed the upregulation of its mRNA and protein localization in seminoma cells. The authors’ prospect is to investigate the exact role of TSPO in normal spermatogenic cell development (from gonocyte to more mature germ cells) and in testicular cancer. The investigation of the TSPO functions takes advantage of the use of synthetic TSPO ligands. Veenman and coworkers contributed to this issue with a review on the role of Tetrapyrroles as endogenous ligands for TSPO in comparison with synthetic ligands [ 22 ]. Interactions between the 18 kDa translocator protein (TSPO) and tetrapyrroles, including the tetrapyrrole protoporphyrin IX 2 Books MDPI Int. J. Mol. Sci. 2017 , 18 , 2640 (PPIX), have been studied for several decades in various species. Thus, Veenman et al. give an overview and the future perspectives for research regarding interactions between TSPO and tetrapyrroles. TSPO can be considered a receptor for PPIX, a transporter for tetrapyrroles, and a participant in the regulation of tetrapyrrole metabolism; vice versa, tetrapyrroles can modulate TSPO functions. A better understanding of the structure–function interactions between TSPO and its endogenous ligands such as tetrapyrroles, including PPIX, may aid in the development of new synthetic TSPO ligands as versatile drugs for the treatment of various diseases. In the review, apart from interactions between TSPO and tetrapyrroles, the effects of synthetic TSPO ligands in the context of TSPO–tetrapyrrole interactions are also presented. Martini and coworkers [ 23 ] contributed here with 13 new high affinity TSPO ligands belonging to their previously described N , N -dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA) class. The new ligands were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The relevance of neurosteroidogenesis in astrocyte well-being was investigated in a human astrocyte model and the positive effect of TSPO-stimulated neurosteroid release on astrocytes well-being was demonstrated. The development of molecules able to stimulate steroid release could represent a therapeutic strategy for central nervous system diseases characterized by astrocyte loss. Furthermore, these ligands may be exploited as pharmacological tools for investigating the autocrine/paracrine roles of neurosteroids in the control of astrocyte metabolism. In order to develop highly selective and active TSPO ligands for cancer therapy and imaging, Lee and coworkers synthesized a new imidazopyridine-based TSPO ligand (CB256) for coordination to 99m Tc and Re [ 24 ]. The 99m Tc-labeled imidazopyridine-based bifunctional chelate ligand was prepared in one step with good radiochemical yield. The resulting complex showed high stability in vitro . The coordination to tricarbonyl rhenium did not alter the TSPO affinity of CB256. In vitro studies on TSPO-rich tumor cells suggested that the radiolabeled complex may have a potential as SPECT radiotracer for the evaluation of TSPO-overexpressing tissues, thus calling for further in vivo biological evaluation. In this special issue Laquintana and coworkers present two TSPO ligand-methotrexate conjugates that are potentially useful for the treatment of TSPO-rich cancers, including brain tumors [ 25 ]. Methotrexate (MTX) is the drug of choice for the treatment of several cancers, but its permeability through the blood–brain barrier (BBB) is poor, making it unsuitable for the treatment of brain tumors. In contrast, the TSPO ligand-MTX conjugates prepared by these authors showed a high binding affinity and selectivity for TSPO, and a more marked toxicity toward glioma cells than MTX alone. These results confirm the ability of the selected TSPO ligand to transport a hydrophilic drug through the biological membranes and determine its accumulation in target cells overexpressing TSPO. The study of Laquintana and coworkers also demonstrates the effectiveness of the bio-conjugate strategy for bringing two agents with a distinct mechanism of action to cancer cells. The use of TSPO ligands for preparing coordination complexes of metallodrugs with diagnostic and/or therapeutic potential has also been exploited by Margiotta and coworkers [ 26 ], who present here the first Pt(IV) derivative of oxaliplatin carrying a ligand for TSPO. This new Pt(IV) complex has been fully characterized from a chemical point of view and has been tested in vitro against human MCF7 breast carcinoma, U87 glioblastoma, and LoVo colon adenocarcinoma cell lines. The affinity for TSPO receptor, the cellular uptake, and the effect on cell cycle progression were also evaluated. The results obtained by these authors render this new coordination complex very promising in the context of a receptor-mediated drug targeting strategy toward TSPO-overexpressing tumors, in particular the colorectal cancer. Finally, Papadopoulos and coworkers contributed to this issue with an article concerning the ability of the peptide VLNYYVW, designed on the TSPO’s CRAC (cholesterol recognition/interaction amino acid consensus) domain, to prevent the opening of the mPTP and the release of apoptotic factors in rat brain mithocondria [ 27 ]. In addition, the authors showed that the TSPO specific drug ligand PK 3 Books MDPI Int. J. Mol. Sci. 2017 , 18 , 2640 11,195 modulates the effects of the CRAC peptide on the induction of mPTP opening and the release of apoptotic factors. These results suggest that TSPO via its C-terminal CRAC domain participates in mPTP function/regulation and apoptosis initiation and that TSPO drug ligands are regulators of this process. 3. Conclusions The high number of papers submitted and ultimately accepted for publication in this special issue attests to the considerable amount of research being conducted on TSPO and TSPO’s role in living cells. TSPO has become an extremely attractive subcellular target for the early detection of disease states (that involve the overexpression of this protein) and for the selective delivery to mitochondria of drugs for diagnostic and therapeutic purposes. Moreover, the effort in the design and synthesis of new, more specific and effective TSPO ligands has been valuable and cannot be neglected. Acknowledgments: We would like to thank all the authors who submitted their work for this special issue. Special thanks also to all of the reviewers who participated and enhanced the quality of the articles by seeking clarification for arguments and requesting modifications where necessary. Conflicts of Interest: The authors declare no conflict of interest. References 1. Biswas, S.; Torchilin, V.P. Nanopreparations for organelle-specific delivery in cancer. Adv. Drug Deliv. Rev. 2014 , 66 , 26–41. [CrossRef] [PubMed] 2. Papadopoulos, V. Structure and function of the peripheral-type benzodiazepine receptor in steroidogenic cells. Proc. Soc. Exp. Biol. Med. 1998 , 217 , 130–142. [CrossRef] [PubMed] 3. Azarashvili, T.; Krestinina, O.; Yurkov, I.; Evtodienko, Y.; Reiser, G. High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca 2+ J. Neurochem. 2005 , 94 , 1054–1062. [CrossRef] [PubMed] 4. Hirsch, J.D.; Beyer, C.F.; Malkowitz, L.; Beer, B.; Blume, A.J. Mitochondrial benzodiazepine receptors mediate inhibition of mitochondrial respiratory control. Mol. Pharmacol. 1989 , 35 , 157–163. [PubMed] 5. Hirsch, T.; Decaudin, D.; Susin, S.A.; Marchetti, P.; Larochette, N.; Resche-Rigon, M.; Kroemer, G. PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection. Exp. Cell Res. 1998 , 241 , 426–434. [CrossRef] [PubMed] 6. Lee, D.H.; Kang, S.K.; Lee, R.H.; Ryu, J.M.; Park, H.Y.; Choi, H.S.; Bae, Y.C.; Suh, K.T.; Kim, Y.K.; Jung, J.S. Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cells. J. Cell. Physiol. 2004 , 198 , 91–99. [CrossRef] [PubMed] 7. Veenman, L.; Papadopoulos, V.; Gavish, M. Channel like functions of the 18-kDa translocator protein (TSPO): Regulation of apoptosis and steroidogenesis as part of the host-defense response. Curr. Pharm. Des. 2007 , 13 , 2385–2405. [CrossRef] [PubMed] 8. Fanizza, E.; Iacobazzi, R.M.; Laquintana, V.; Valente, G.; Caliandro, G.; Striccoli, M.; Agostiano, A.; Cutrignelli, A.; Lopedota, A.; Curri, M.L.; et al. Highly selective luminescent nanostructures for mitochondrial imaging and targeting. Nanoscale 2016 , 14 , 3350–3361. [CrossRef] [PubMed] 9. Sekimata, K.; Hatano, K.; Ogawa, M.; Abe, J.; Magata, Y.; Biggio, G.; Serra, M.; Laquintana, V.; Denora, N.; Latrofa, A.; et al. Radiosynthesis and in vivo evaluation of N -[ 11 C]methylated imidazopyridineacetamides as PET tracers for peripheral benzodiazepine receptors. Nucl. Med. Biol. 2008 , 35 , 327–334. [CrossRef] [PubMed] 10. Denora, N.; Laquintana, V.; Lopalco, A.; Iacobazzi, R.M.; Lopedota, A.; Cutrignelli, A.; Iacobellis, G.; Annese, C.; Cascione, M.; Leporatti, S.; et al. In vitro targeting and imaging the translocator protein TSPO 18-kDa through G(4)-PAMAM-FITC labeled dendrimer. J. Control. Release 2013 , 172 , 1111–1125. [CrossRef] [PubMed] 11. Denora, N.; Cassano, T.; Laquintana, V.; Lopalco, A.; Trapani, A.; Cimmino, C.S.; Laconca, L.; Giuffrida, A.; Trapani, G. Novel codrugs with GABAergic activity for dopamine delivery in the brain. Int. J. Pharm. 2012 , 437 , 221–231. [CrossRef] [PubMed] 4 Books MDPI Int. J. Mol. Sci. 2017 , 18 , 2640 12. Denora, N.; Laquintana, V.; Trapani, A.; Lopedota, A.; Latrofa, A.; Gallo, J.M.; Trapani, G. Translocator protein (TSPO) ligand-Ara-C (cytarabine) conjugates as a strategy to deliver antineoplastic drugs and to enhance drug clinical potential. Mol. Pharm. 2010 , 7 , 2255–2269. [CrossRef] [PubMed] 13. Johnstone, T.C.; Suntharalingam, K.; Lippard, S.J. The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016 , 116 , 3436–3486. [CrossRef] [PubMed] 14. Laquintana, V.; Denora, N.; Lopalco, A.; Lopedota, A.; Cutrignelli, A.; Lasorsa, F.M.; Agostino, G.; Franco, M. Translocator protein ligand-PLGA conjugated nanoparticles for 5-fluorouracil delivery to glioma cancer cells. Mol. Pharm. 2014 , 11 , 859–871. [CrossRef] [PubMed] 15. Denora, N.; Iacobazzi, R.M.; Natile, G.; Margiotta, N. Metal complexes targeting the Translocator Protein 18 kDa (TSPO). Coord. Chem. Rev. 2017 , 341 , 1–18. [CrossRef] 16. Iacobazzi, R.M.; Lopalco, A.; Cutrignelli, A.; Laquintana, V.; Lopedota, A.; Franco, M.; Denora, N. Bridging Pharmaceutical Chemistry with Drug and Nanoparticle Targeting to Investigate the Role of the 18-kDa Translocator Protein TSPO. ChemMedChem 2017 , 12 , 1261–1274. [CrossRef] [PubMed] 17. Awad, M.; Gavish, M. Binding of [ 3 H]Ro 5-4864 and [ 3 H]PK 11195 to cerebral cortex and peripheral tissues of various species: species differences and heterogeneity in peripheral benzodiazepine binding sites. J. Neurochem. 1987 , 49 , 1407–1414. [CrossRef] [PubMed] 18. Wohlrab, H. Transport proteins (carriers) of mitochondria. IUBMB Life 2009 , 61 , 40–46. [CrossRef] [PubMed] 19. Giudetti, A.M.; Stanca, E.; Siculella, L.; Gnoni, G.V.; Damiano, F. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism. Int. J. Mol. Sci. 2016 , 17 , 817. [CrossRef] [PubMed] 20. Yasin, N.; Veenman, L.; Singh, S.; Azrad, M.; Bode, B.; Vainshtein, A.; Caballero, B.; Marek, I.; Gavish, M. Classical and Novel TSPO Ligands for the Mitochondrial TSPO Can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling. Int. J. Mol. Sci. 2017 , 18 , 786. [CrossRef] [PubMed] 21. Manku, G.; Culty, M. Regulation of Translocator Protein 18 kDa (TSPO)Expression in Rat and Human Male Germ Cells. Int. J. Mol. Sci. 2016 , 17 , 1486. [CrossRef] [PubMed] 22. Veenman, L.; Vainshtein, A.; Yasin, N.; Azrad, M.; Gavish, M. Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands. Int. J. Mol. Sci. 2016 , 17 , 880. [CrossRef] [PubMed] 23. Da Pozzo, E.; Giacomelli, C.; Costa, B.; Cavallini, C.; Taliani, S.; Barresi, E.; Da Settimo, F.; Martini, C. TSPO PIGA Ligands Promote Neurosteroidogenesis and Human AstrocyteWell-Being. Int. J. Mol. Sci. 2016 , 17 , 1028. [CrossRef] [PubMed] 24. Choi, J.Y.; Iacobazzi, R.M.; Perrone, M.; Margiotta, N.; Cutrignelli, A.; Jung, J.H.; Park, D.D.; Moon, B.S.; Denora, N.; Kim, S.E.; et al. Synthesis and Evaluation of Tricarbonyl 99m Tc-Labeled 2-(4-Chloro)phenyl-imidazo[1,2-a] pyridine Analogs as Novel SPECT Imaging Radiotracer for TSPO-Rich Cancer. Int. J. Mol. Sci. 2016 , 17 , 1085. [CrossRef] [PubMed] 25. Valentino Laquintana, V.; Denora, N.; Cutrignelli, A.; Perrone, M.; Iacobazzi, R.M.; Annese, C.; Lopalco, A.; Lopedota, A.A.; Franco, M. TSPO Ligand-Methotrexate Prodrug Conjugates: Design, Synthesis, and Biological Evaluation. Int. J. Mol. Sci. 2016 , 17 , 967. [CrossRef] [PubMed] 26. Savino, S.; Denora, N.; Iacobazzi, R.M.; Porcelli, L.; Azzariti, A.; Natile, G.; Margiotta, N. Synthesis, Characterization, and Cytotoxicity of the First Oxaliplatin Pt(IV) Derivative Having a TSPO Ligand in the Axial Position. Int. J. Mol. Sci. 2016 , 17 , 1010. [CrossRef] [PubMed] 27. Azarashvili, T.; Krestinina, O.; Baburina, Y.; Odinokova, I.; Akatov, V.; Beletsky, I.; Lemasters, J.; Papadopoulos, V. Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria. Int. J. Mol. Sci. 2016 , 17 , 2096. [CrossRef] [PubMed] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 5 Books MDPI International Journal of Molecular Sciences Review Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism Anna M. Giudetti, Eleonora Stanca, Luisa Siculella *, Gabriele V. Gnoni and Fabrizio Damiano Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce 73100, Italy; anna.giudetti@unisalento.it (A.M.G.); eleonora.stanca@unisalento.it (E.S.); gabriele.gnoni@unisalento.it (G.V.G.); fabrizio.damiano@unisalento.it (F.D.) * Correspondence: luisa.siculella@unisalento.it; Tel.: +39-8-3229-8696 Academic Editors: Giovanni Natile and Nunzio Denora Received: 13 April 2016; Accepted: 19 May 2016; Published: 25 May 2016 Abstract: The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism. Keywords: β -oxidation; carnitine/acylcarnitine translocase; citrate carrier; fatty acid synthesis; hormonal regulation; nutritional regulation 1. Introduction Mitochondria are well-defined cytoplasmic organelles, which undertake multiple critical functions in the cell. In addition to oxidative phosphorylation (OXPHOS), a pathway in which nutrients are oxidized to form adenosine triphosphate (ATP), mitochondria are involved in several pathways including citric acid cycle, gluconeogenesis, fatty acid oxidation and lipogenesis, amino acid degradation and heme biosynthesis. They produce most of the cellular reactive oxygen species (ROS), buffer cellular Ca 2+ and they initiate cellular apoptosis [ 1 – 3 ]. Moreover, mitochondria participate in cell communication and inflammation, and play an important role in aging, drug toxicity, and pathogenesis [4]. Mitochondria and cytosol are engaged in numerous metabolic processes which, due to enzyme compartmentalization, involve the exchange of metabolites among them. Energy transduction in mitochondria requires the transport of specific metabolites across the inner membrane, achieved through mitochondrial carriers (MCs), a family of nuclear-encoded proteins sharing several structural features. Their common function is to provide a link between mitochondria Int. J. Mol. Sci. 2016 , 17 , 817 6 www.mdpi.com/journal/ijms Books MDPI Int. J. Mol. Sci. 2016 , 17 , 817 and cytosol by facilitating the flux of a high number of metabolites through the permeability barrier of the inner mitochondrial membrane (IMM). In humans, MCs are encoded by the SLC25 genes, and some of them have isoforms encoded by different genes [ 4 ]. Until now, 53 mitochondrial carriers have been identified on the human genome and more than half have been functionally characterized [4]. Members of SLC25 family, mainly located in mitochondria, have been found in all eukaryotes; few of them are present in peroxisomes and chloroplasts [ 5 ]. SLC25 genes are highly variable in size and organization, whereas their products are very similar sharing a tripartite structure composed by 100 amino acid repeats [4]. The complete sequence analysis of some MCs during the late 1990s showed that each domain contains two transmembrane α -helices, separated by hydrophilic regions, and a common signature motif which can be divided into a first part, P-X-D/E-X-X-K/R, and a second part as [D/E]GXXXX[W/Y/F][R/K]G [ 4 ]. The common structure is reflected in a similar function. To transport solutes across IMM, many MCs catalyze an exchange reaction: they have only one binding site, which is alternately exposed to the two opposite sides of the membrane. The substrate-induced conformational changes occur during the transition from cytosol to matrix and vice versa [ 4 ]. However, MCs do not adopt only antiport as transport; the carnitine-acylcarnitine translocase (CACT) catalyzes both unidirectional transport of carnitine and the carnitine/acylcarnitine exchange [ 6 ], whereas the uncoupling protein catalyzes uniport as the exclusive transport mode [7]. Functional characterization of MCs was carried out through their purification and reconstitution in artificial membranes, such as proteoliposomes. Studies on the transport activity performed with the liposomal systems show a dependence of the kinetic parameters on the lipid composition of the mitochondrial membrane, particularly on the cardiolipin (CL) levels [ 8 ]. CL interacts with a number of proteins and enzymes involved in fundamental mitochondrial bioenergetic processes [ 9 ]. Thus, CL is crucial for mitochondrial OXPHOS and for correct structure and function of IMM. It has been proposed that CL creates an environment protecting and stabilizing MCs in a functionally intact state [8]. MCs play a crucial role in intermediary metabolism. In this respect, citrate carrier (CiC) and CACT are two MCs mainly involved in fatty acid metabolism. CiC, encoded by SLC25A1 , promotes the efflux of citrate from the mitochondria to the cytosol where citrate is cleaved by ATP-citrate lyase to oxaloacetate (OAA) and acetyl-coenzyme A (acetyl-CoA), which is used for fatty acid and sterol syntheses. CACT catalyzes the transport of fatty acids, in the form of acylcarnitine, into mitochondria, where they are oxidized by the enzymes of β -oxidation pathway (Figure 1). These MCs are mutually regulated to allow the synthesis and oxidation of fatty acids to take place at different times. The aim of this review is to summarize biochemical, molecular, and physio-pathological aspects of CiC and CACT. 7 Books MDPI Int. J. Mol. Sci. 2016 , 17 , 817 Figure 1. Schematic model of citrate carrier (CiC) and carnitine-acylcarnitine translocase (CACT) in lipogenesis and β -oxidation, and their metabolic interrelationship. Abbreviations: ACC, acetyl-CoA carboxylase; CoA-SH, coenzyme-A; CPT1, carnitine palmitoyltransferase 1; CPT2, carnitine palmitoyltransferase 2; DN L, de novo lipogenesis; FAS, fatty acid synthase; IMM, inner mitochondrial membrane; IMS, intermembrane space; OAA, oxaloacetate; OMM, outer mitochondrial membrane; PFK-1, phosphofructokinase-1. The green arrow and dashed red lines represent, respectively, positive (+) and negative (-) allosteric modulation of the indicated target enzymes. 2. Citrate Carrier (CiC) and Carnitine-Acylcarnitine Translocase (CACT): Mitochondrial Carriers in Fatty Acid Metabolism Fatty acids perform several functions in cells; as components of triacylglycerols, they represent the main form of stored energy, and as constituents of phospholipids they play important structural roles, while some of them are also involved in intracellular signaling. Fatty acid metabolism requires the involvement of both cytosolic and mitochondrial reactions (Figure 1). The de novo lipogenesis ( DN L) ( i.e. , de novo fatty acid synthesis) takes place in the cytosol. The initiation of DN L occurs in the presence of high levels of blood glucose, indicating a sufficient energy intake. In this condition, the pancreas secretes insulin, which not only promotes the uptake of glucose from blood into the cells but also stimulates the synthesis of two enzymes of the DN L, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) [ 10 ]. These enzymes work in sequence to convert first acetyl-CoA to malonyl-CoA, in a reaction catalyzed by ACC, and then, by a series of reactions catalyzed by FAS, to produce palmitate, a saturated fatty acid with 16 carbon atoms. The condensation of malonyl, bound to acyl carrier protein (ACP), with acetyl-CoA by the ketoacyl-ACP synthase, is the first reaction catalyzed by FAS. Acetyl-CoA utilized for DN L is mainly derived from carbohydrate metabolism. In this respect, after glucose conversion into pyruvate, the latter enters through its specific transporter into mitochondria, where it is converted in acetyl-CoA in a reaction catalyzed by pyruvate dehydrogenase. In the Krebs cycle, acetyl-CoA is then converted into citrate (tricarboxylate) after condensation with OAA. In a good energetic state, citrate is transported into the cytosol via CiC in exchange for malate (dicarboxylate) (the citrate/malate antiporter). The exchange is electroneutral being citrate efflux 8 Books MDPI Int. J. Mol. Sci. 2016 , 17 , 817 compensated by a contemporary efflux of a proton [ 4 ]. CiC transport activity is particularly high in the liver where active fatty acid synthesis occurs, and it is virtually absent in other tissues. CiC mRNA and/or protein levels are high in the liver, pancreas, and kidney, but are low or absent in the brain, heart, skeletal muscle, placenta, and lungs [11]. By the action of ATP-citrate lyase, cytosolic citrate is converted into OAA and acetyl-CoA, and this latter is used for the synthesis of fatty acids and cholesterol. OAA produced in the cytosol by ATP-citrate lyase is reduced to malate, which is converted to pyruvate via the malic enzyme with production of cytosolic NADPH plus H + necessary for fatty acid and sterol syntheses. More